Combined in silico and experimental identification of the Pyrococcus abyssi H/ACA sRNAs and their target sites in ribosomal RNAs

ABSTRACT Motivation Clustered regularly interspaced short palindromic repeats (CRISPR) technologies allow for facile genomic modification in a site-specific manner. A key step in this process is the in silico design of single guide RNAs to efficiently and specifically target a site of interest. To this end, it is necessary to enumerate all potential off-target sites within a given genome that could be inadvertently altered by nuclease-mediated cleavage. Currently available software for this task is limited by computational efficiency, variant support or annotation, and assessment of the functional impact of potential off-target effects. Results To overcome these limitations, we have developed CRISPRitz, a suite of software tools to support the design and analysis of CRISPR/CRISPR-associated (Cas) experiments. Using efficient data structures combined with parallel computation, we offer a rapid, reliable, and exhaustive search mechanism to enumerate a comprehensive list of putative off-target sites. As proof-of-principle, we performed a head-to-head comparison with other available tools on several datasets. This analysis highlighted the unique features and superior computational performance of CRISPRitz including support for genomic searching with DNA/RNA bulges and mismatches of arbitrary size as specified by the user as well as consideration of genetic variants (variant-aware). In addition, graphical reports are offered for coding and non-coding regions that annotate the potential impact of putative off-target sites that lie within regions of functional genomic annotation (e.g. insulator and chromatin accessible sites from the ENCyclopedia Of DNA Elements [ENCODE] project). Availability and implementation The software is freely available at: https://github.com/pinellolab/CRISPRitzhttps://github.com/InfOmics/CRISPRitz. Supplementary information Supplementary data are available at Bioinformatics online.

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IN SILICO ANALYSIS OF POTENTIAL OFF-TARGET SITES TO GENE EDITING FOR MUCOPOLYSACCHARIDOSIS TYPE I BY USING THE CRISPR/CAS9 SYSTEM: IMPLICATIONS FOR POPULATIONS-SPECIFIC TREATMENTS

Cytotherapy ◽

10.1016/j.jcyt.2021.02.057 ◽

2021 ◽

Vol 23 (4) ◽

pp. 19

Author(s):

PB Carneiro ◽

MV Freitas ◽

US Matte

Keyword(s):

In Silico ◽

Gene Editing ◽

In Silico Analysis ◽

Type I ◽

Mucopolysaccharidosis Type ◽

Mucopolysaccharidosis Type I ◽

Target Sites ◽

Silico Analysis

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An in silico approach to identify and prioritize miRNAs target sites polymorphisms in colorectal cancer and obesity

Cancer Medicine ◽

10.1002/cam4.3546 ◽

2020 ◽

Vol 9 (24) ◽

pp. 9511-9528

Author(s):

Morteza Gholami ◽

Marzieh Zoughi ◽

Bagher Larijani ◽

Mahsa M. Amoli ◽

Milad Bastami

Keyword(s):

Colorectal Cancer ◽

In Silico ◽

Target Sites

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Targeted and genome-wide sequencing reveal single nucleotide variations impacting specificity of Cas9 in human stem cells

Nature Communications ◽

10.1038/ncomms6507 ◽

2014 ◽

Vol 5 (1) ◽

Cited By ~ 87

Author(s):

Luhan Yang ◽

Dennis Grishin ◽

Gang Wang ◽

John Aach ◽

Cheng-Zhong Zhang ◽

...

Keyword(s):

Stem Cells ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

In Silico ◽

High Efficiency ◽

Specific Gene ◽

Whole Genome ◽

Single Nucleotide ◽

Target Sites ◽

Target Effects

Abstract CRISPR/Cas9 has demonstrated a high-efficiency in site-specific gene targeting. However, potential off-target effects of the Cas9 nuclease represent a major safety concern for any therapeutic application. Here, we knock out the Tafazzin gene by CRISPR/Cas9 in human-induced pluripotent stem cells with 54% efficiency. We combine whole-genome sequencing and deep-targeted sequencing to characterise the off-target effects of Cas9 editing. Whole-genome sequencing of Cas9-modified hiPSC clones detects neither gross genomic alterations nor elevated mutation rates. Deep sequencing of in silico predicted off-target sites in a population of Cas9-treated cells further confirms high specificity of Cas9. However, we identify a single high-efficiency off-target site that is generated by a common germline single-nucleotide variant (SNV) in our experiment. Based on in silico analysis, we estimate a likelihood of SNVs creating off-target sites in a human genome to be ~1.5–8.5%, depending on the genome and site-selection method, but also note that mutations might be generated at these sites only at low rates and may not have functional consequences. Our study demonstrates the feasibility of highly specific clonal ex vivo gene editing using CRISPR/Cas9 and highlights the value of whole-genome sequencing before personalised CRISPR design.

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